Purified Antigen for Alzheimer&#39;s Disease and Methods of Obtaining and Using Same

ABSTRACT

The invention relates, among other things, a preparation comprising Alzheimer&#39;s disease antigen (A68), as well as methods of obtaining this purified antigen, and methods of using this purified antigen, for instance, for diagnosing Alzheimer&#39;s disease and for detecting human autoantibodies to the Alzheimer disease antigen. The antigen preparation according to the invention is purified in that it is substantially free of immunoglobulin G. The invention further relates to methods of making Alzheimer disease antigens that can be used instead of or along with the A68 antigen preparation (e.g., for diagnosing AD), such as recombinant human tau, tau isolated from various species including human, and phosphorylated recombinant human tau or isolated tau, as well as A68 anti-idiotypic antibodies.

TECHNICAL FIELD OF THE INVENTION

The invention relates to a preparation comprising Alzheimer's diseaseantigen (A68), to methods of obtaining this purified antigen, and tomethods of using this purified antigen preparation, for instance, indiagnosing Alzheimer's disease. The antigen preparation according to theinvention is purified in that it is substantially free of immunoglobulinG. The invention further relates to methods of making Alzheimer diseaseantigens that can be used instead of or along with the A68 antigenpreparation (e.g., for diagnosing AD), such as recombinant human tau,tau isolated from various species including human, and phosphorylatedrecombinant human tau or isolated tau, as well as A68 anti-idiotypicantibodies.

BACKGROUND OF THE INVENTION

Alzheimer's disease (AD) is a progressive neurodegenerative disorderaffecting 7% of the population over 65 years of age and characterizedclinically by progressive loss of intellectual function andpathologically by a continuing loss of neurons from the cerebral cortex.This pathological impairment usually is correlated with increasednumbers of neuritic plaques in the neocortex and with the loss ofpresynaptic markers of cholinergic neurons. Neuritic plaques arecomposed of degenerating axons and nerve terminals, as well as possibleastrocytic elements, and these plaques often exhibit a central amyloidcore.

Another characteristic pathological feature of Alzheimer's disease isdevelopment of neurofibrillary tangles. A neurofibrillary tangle is anintraneuronal mass composed of normal intermediate filaments and pairedhelical filaments having unusual properties, which twist and formtangles. Neurofibrillary tangles are comprised of several differentproteins.

Neurochemical studies confirm that neurotransmitter systems aredeleteriously affected by Alzheimer's disease. The most consistently andseverely affected system is that of the cholinergic neurons located inthe Nucleus Basalis of Meynert. In addition, a reduction insomatostatin, substance P, and corticotropin releasing factor areobserved.

None of the above-mentioned pathologic states such as neurochemicalalterations, neuritic plaques or neurofibrillary tangles are unique toAlzheimer's disease. These impairments also occur in the brains ofnormal aged individuals and are associated with other diseases such asGuam Parkinson's Disease, Dementia Pugilistica and ProgressiveSupra-nuclear Palsy. For example, paired helical filaments, the twistedfilaments that form the tangles and fill the neurites of plaques, alsooccur in certain other diseases. In fact, immunologic studies have shownthat AD epitopes of paired helical filaments exist in Pick bodies, thespherical structures found in affected neurons in the temporal cortex ofbrains affected by Pick's Disease. In addition, the densities ofneurofibrillary tangles and neuritic plaques within the cerebral cortexof an Alzheimer's disease patient correlates only weakly with the stagesof the illness.

Accordingly, the diagnosis of Alzheimer's disease has been extremelydifficult. Ante-mortem diagnosis of the disease is performed primarilyby exclusion of other diseases. An article entitled, “The Neurochemistryof Alzheimer's Disease and Senile Dementia”, by Peter Davies inMedicinal Research Reviews, Vol. 3, No. 3, pp. 221-236 (1983), discussesAlzheimer's disease and at page 223 states:

-   -   The problem in the diagnosis of Alzheimer's disease is that        there is no positive test: the clinician has to rule out other        causes of dementia such as strokes, microvascular disease, brain        tumors, thyroid dysfunction, drug reactions, severe depression        and a host of other conditions that can cause intellectual        deficits in elderly people. Only when all of these problems have        been eliminated as a cause of the symptoms should a diagnosis of        Alzheimer's disease be accepted.

Post-mortem diagnosis of Alzheimer's disease has been based ondetermination of the number of neuritic plaques and tangles in braintissue using specialized staining techniques. However, such diagnosticmethods, based on neurohistopathological studies, require extensivestaining and microscopic examination of several brain sections.Moreover, the plaques and tangles are not confined to individuals havingAlzheimer's disease, but also may occur in the brains of normal, elderlyindividuals or individuals with other diseases. Thus, a more definitiveand reliable method for making the diagnosis is needed.

U.S. Pat. No. 4,666,829 issued to Glenner et al. discloses attempts toidentify an antigen specific for Alzheimer's disease. However, theantigen described by Glenner et al. also is present in adults ofadvanced age who do not have Alzheimer's disease (see Ghanbari et al.,Journal of the American Medical Association, 263, pp. 2907-2910 (1990)).Therefore, a need still exists for a method of diagnosing Alzheimer'sdisease as distinct from other diseases or age-related indicia.

Similarly, U.S. Pat. No. 5,492,812 issued to Voorheis et al. describes adiagnostic method for Alzheimer's disease that is carried out byscreening for tau peptides in the blood of a patient. This method callsfor the use of an antibody or Fab fragment that specifically binds taupeptide derived from either the amino terminal 200 amino acids orcarboxy terminal 50 amino acids of a tau protein. This method requiresthat “the whole of the 200 amino acid N-terminal residues of the varioustau proteins as well as some portion of their 50 amino acid mostC-terminal residues will be released when cleaved from the filaments byubiquitin-recognizing proteases or other proteases during degenerationand rupture of the affected neurons” (col. 5, lines 12-19). The methodfurther requires that the cleaved segments find their way into bodyfluids outside the brain (col. 5, lines 19-22). Accordingly, the methodis dependent upon, and is ineffective in the absence of, proteolyticfragmentation of the tau complex of proteins. The method further isdependent upon, and is ineffective in the absence of, the subsequentrelease of the proteolytic fragments into body fluids. It thus isdesirable that a direct means of assay for Alzheimer disease-associatedantigens be identified, particularly a means that does not requireproteolytic fragmentation and subsequent release into the bloodstream offragments.

Along these lines, PCT International Application WO 96/20218 of Ghanbariet al. describes the isolation of an antigen associated with Alzheimer'sdisease and a monoclonal antibody directed against this antigen. Thisantigen is specific for Alzheimer's disease, being present in highquantities in Alzheimer's Disease patients, and being nearlynon-detectable in non-Alzheimer's Disease patients. However, the antigenis described as being only “partially purified” in the preparation ofGhanbari et al., consisting of an aggregate of proteins, with thepredominant protein having a molecular weight of about 68,000 daltons,and including tau and hyperphosphorylated tau (see, e.g, Example 2).Accordingly, for some applications, a more purified preparation of thisAlzheimer's Disease antigen may be desirable and/or required.

It therefore is an object of this invention to provide, among otherthings, a purified preparation of an Alzheimer's disease antigen. It isanother object of this invention to provide, among other things, amethod of diagnosing Alzheimer's disease using the purified preparation.It is a further object of this invention to provide, methods ofobtaining the purified preparation of the Alzheimer's disease antigen.These and other objects and advantages of the present invention, as wellas additional inventive features, will be apparent from the descriptionof the invention provided herein.

BRIEF SUMMARY OF THE INVENTION

The invention provides, among other things, a preparation comprisingAlzheimer's disease antigen (A68), as well as methods of obtaining thispurified antigen (Ag), and methods using the purified Ag, for instance,for diagnosing Alzheimer's Disease (AD). This Ag is purified in that itis substantially free of immunoglobulin G (IgG). The inventionadditionally provides methods of making AD Ags that can be used insteadof or along with A68 (e.g., for binding AD autoantibodies), such asrecombinant human tau, tau isolated from various species includinghuman, and phosphorylated recombinant human tau or isolated tau, as wellas A68 anti-idiotype antibodies (Abs). The invention further describestreatments of these Ags that enhance their reactivity withautoantibodies directed against A68. These treatments include treatmentwith hypericin, free fatty acids, and/or hydroxynonenal or otheradvanced glycation end products.

The invention also describes methods using a bovinemicrotubule-associated protein preparation (MAPf) for diagnosing AD. Theinvention describes analysis of autoantibody reactivity with both A68and MAPf such that either a quantitative or a qualitative analysis ofthese reactivities provides a diagnosis for AD.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a reproduction of a photograph of a Western blot for detectionof anti-A68 autoantibodies in Alzheimer's disease patients where thestrips were probed with sera from two Alzheimer's disease patients(Patient 1, lane 2; Patient 2, lane 4) and two normal controls (Patient3, lane 3; Patient 4, lane 5). Relative molecular weights are listed tothe left of the blot. Lane 1 demonstrates the A68 banding patternproduced by an anti-A68 monoclonal antibody.

FIG. 2 is a reproduction of a photograph of a Western blot for detectionof anti-A68 autoantibodies in Alzheimer's disease patients using twolots of partially purified A68 preparation (“Lot A”, lanes 1-3, and “LotB”, lanes 4-6) and one lot of Protein A/G treated A68 preparation (lanes7-9). The strips were probed with sera from two Alzheimer's diseasepatients (Patient 1, lanes 2, 5, 8 or Patient 2, lanes 3, 6, 9) followedby goat anti-human IgG-HRP, or the strips were probed for IgG endogenousto the A68 preparations using the goat anti-human HRP antibody only(lanes 1, 4, 7). Symbols: Arrow, ˜55 kD, shows the heavy chain of IgG;Arrowhead, shows the prominent A68 bands.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

By “individuals with Alzheimer's disease” is meant individuals orpatients suffering from, affected by, or manifesting the clinicalsymptoms of the disease. According to the invention, a diagnosis of“Alzheimer's disease” is based on accepted standards of clinicaldiagnosis. Preferably, an individual according to the inventiondiagnosed with Alzheimer's disease is one that has met most (i.e., amajority) of the generally accepted criteria for diagnosis ofAlzheimer's disease. Desirably, because of certain atypical features,such an individual may be described by a physician as ‘possibleAlzheimer's disease’, and at least 75% to 85% of these individuals wouldbe found at autopsy to have suffered from Alzheimer's disease. Even morepreferably, an individual according to the invention diagnosed withAlzheimer's disease is one that has met the best available clinicalcriteria for the diagnosis of Alzheimer's disease, and may be describedby a physician as “probable Alzheimer's disease”. When such a diagnosisis made by one skilled in the art, optimally about 90% of these patientswould be found on autopsy to have Alzheimer's disease.

Alzheimer's Disease Antigen Preparation

The present invention pertains, inter alia, to the Alzheimer's diseaseantigen designated A68. The antigen has been described as obtained fromthe brains and cerebrospinal fluid (CSF) of Alzheimer patients, but isreferred to herein as the “Alzheimer antigen” regardless of where it isfound in a person if the antigen has the properties set forth herein. Ithas been discovered that one such Alzheimer antigen is a protein, sothat the Alzheimer antigen is also referred to herein as an “Alzheimerprotein” when the protein property is a prominent factor in thediscussion. The autoantibodies which are immunologically reactive withthe Alzheimer antigen are referred to herein as “Alzheimer antibodies.”The Alzheimer's disease antigen also refers to components of apreparation containing this antigen that have been modified to increasetheir reactivity with antibodies directed against the antigen, asfurther described below.

The identification, isolation, and characterization of the Alzheimer'sdisease antigen as a “partially purified preparation of A68 antigen” hasbeen described in PCT International Application WO 96/20218 (see, e.g.,Example 2 in particular, as well as remainder of this document which isincorporated in its entirety by reference). In particular, PCTInternational Application WO 96/20218 describes the confinement of thisantigen to Alzheimer's disease patients (see, Example 2A, Example 8) ascompared to normal individuals, and individuals having otherimpairments. Generally, the Alzheimer antigen is found in Alzheimerpatients while being present in much reduced (or non-measurable)quantities in non-Alzheimer patients, including patients suffering fromother neurologic diseases.

The antigen of the invention which is associated with Alzheimer'sdisease (i.e., the Alzheimer's antigen) is an aggregate of severalproteins, and the major protein species have an apparent molecularweight of about 68,000 daltons on a reducing SDS gel. The aggregatemigrates electrophoretically as a band or bands on sodium dodecylsulfate polyacrylamide gel with an apparent M, of from about 60 to about70 kDa. A68 desirably is prepared from human brain (typically frozen),most often from cerebral cortex from an Alzheimer's disease patient.Other properties of the A68 antigen are set forth in PCT Internationalapplication WO 96/20218, and include, but are not limited to, thefollowing characteristics: immunologically reactive with a monoclonalantibody produced by the hybridoma cell line identified as ATCC No.HB9205 (i.e., ALZ-50, further described below); has an isoelectric pointof about 6 in reduced or non-reduced form; binds to an affi-Blue column;is at least 50% soluble in a solution of 0.01 M sodium phosphate, 0.14 Msodium chloride and 1 mM phenyl methyl sulfonyl fluoride at pH 6.8, andprecipitates in 50% saturated ammonium sulfate at 4° C.

Since its first description, the Alzheimer's antigen has beenadditionally referred to as A68, tau, hyperphosphorylated tau (Lee etal., Science 251: 675-678, 1991), abnormally phosphorylated tau(Grundke-Iqbal et al., Proc. Natl. Acad. Sci. 83: 4913-4917, 1986),soluble PHF (Greenberg and Davies, Proc. Natl. Acad. Sci. 87: 5827-5831,1990), PHF tau (Greenberg et al., J. Biol. Chem. 267: 564-569, 1992),and Alzheimer's Disease Associated Protein (ADAP) (Ghanbari et al., JAMA263: 2907-2910, 1990). All terms are deemed to be equivalent whenreferring to the Alzheimer's antigen herein. It contains tau andphosphorylated tau. Thus, according to the invention, A68 refers to aform of the microtubule-associated protein tau which, in Alzheimer'sdisease, is the primary protein constituent of paired helical filaments.Relative to normal tau (also a microtubule protein), it ishyperphosphorylated and exhibits an altered conformation.

A68 is obtained in only a “partially purified” form as described inExample 1 of PCT International Application WO 96/20218. By comparison,as described herein, in the process of obtaining a purified preparationof A68 antigen, it was discovered that, not only did the preparationcomprise expected elements that needed to be removed (e.g., proteins,etc.), but also, that the preparation surprisingly containedimmunoglobulin G (IgG). Removal of such IgG substantially andsurprisingly increases the effectiveness of the antigen preparation, andits effectiveness for use in an assay.

Accordingly, the present invention provides a protein preparationconsisting essentially of an antigen that is immunologically reactivewith a monoclonal antibody produced by the hybridoma cell lineidentified as ATCC No. HB9205 (i.e., Alz-50), wherein the preparation issubstantially free of immunoglobulin G. As used herein, “substantiallyfree of immunoglobulin G” means a preparation having an amount ofimmunoglobulin G that preferably is equal to or less than about 0.05% ofthe total protein present in the preparation, and even more preferablyis less than or equal to about 0.0015% of the total protein present inthe preparation, and/or a total immunoglobulin amount that desirably isless than about 500 pg of immunoglobulin G per μg of A68 (i.e., when A68amount is assessed by Western analysis with use of the Alz-50 antibody,or by other appropriate means), and optimally is less than about 15 pgof immunoglobulin G per μg of A68. In particular, by substantially freeof immunoglobulin G desirably is meant a level of immunoglobulin thatdoes not interfere with the assays of the invention.

Further, the present invention desirably provides a purified preparationof antigen, which preparation is a diagnostic marker of Alzheimer'sdisease, wherein the antigen preferably comprises a major polypeptidespecies that:

(a) has an isoelectric point of about 6 in reduced or non-reduced form;

(b) binds to an affi-Blue column;

(c) is at least 50% soluble in a solution of 0.01 M sodium phosphate,0.14 M sodium chloride and 1 mm phenyl methyl sulfonyl fluoride (PMSF)at pH 6.8, and precipitates in 50% saturated ammonium sulfate at 4° C.;

(d) is immunologically reactive with a monoclonal antibody produced bythe hybridoma cell line identified as ATCC No. HB9205 (i.e., Alz-50);and

(e) is substantially free of immunoglobulin G (i.e., as describedabove).

Process For Obtaining an Alzheimer Antigen Protein Preparation

The present invention thus provides a process for obtaining an Alzheimerantigen protein preparation. As a first step, desirably a partiallypurified preparation is obtained. A partially purified A68 preparationdesirably is obtained by a method that comprises:

(a) obtaining a sample of cortical brain tissue containing the antigen;

(b) homogenizing the sample in buffer to obtain a homogenate;

(c) removing particulate matter from the homogenate;

(d) removing the Ag from the homogenate by contacting the homogenatewith an antibody (i.e., immobilized AD antibody, as in affinitypurification) under conditions wherein the antigen and antibody bind toform an antigen-antibody complex; and

(e) eluting the antigen from the antigen-antibody complex to obtain apartially purified protein preparation.

More specifically, this method of preparation desirably is carried outby homogenization of tissue in about 5 volumes of an aqueous buffer suchas Tris buffered saline (TBS), preferably where the buffer furthercontains protease and phosphatase inhibitors. The homogenate optimallyis fractionated, e.g., by centrifugation at about 27,000×g for about 60minutes at about 4° C. Desirably, the supernatant is collected, andpassed over an affinity column, preferably in an iterative fashion forabout 16 hours at about 4° C. Optimally the affinity column is a MC1column, desirably which is prepared by using a purified mouse monoclonalantibody (e.g., MC1 antibody, as described in the Examples) which reactsspecifically with A68, and coupling the antibody to Affigel-10,according to manufacturer's instructions. Preferably, A68 isspecifically removed from the supernatant by attaching to the MC1 columnmatrix. Following extensive washing with TBS, optimally A68 is elutedfrom the MC1 column using, for instance, 3 M KSCN. The A68 preparationpreferably is subsequently dialyzed against buffer (e.g., TBS) andstored at −80° C. Alternative means of obtaining partially purified A68preparations are set out, for example, in PCT International ApplicationWO 96/20218.

The preparation obtained by this process is highly enriched in A68, butcontains amounts of other proteins. In particular, this preparationcontains endogenous human immunoglobulins that comprise approximatelyfrom about 1 to about 5% of the total protein. These immunoglobulinsinterfere with the ability to detect serum autoantibodies to A68 byeither Western analysis (e.g.; FIG. 2, Examples) or ELISA (e.g., Table2, Examples). Such an A68 protein preparation is not substantially pureaccording to the invention, but is only partially purified.

Thus, it is necessary according to the invention to remove thecontaminating immunoglobulins prior to analysis of serum autoantibodies,or prior to use of the A68 protein preparation. The inventionaccordingly provides an additional step (f) in the process describedabove which step comprises removing immunoglobulin G from the eluent toobtain the antigen preparation that is substantially free ofimmunoglobulin G. This is accomplished, for instance, by incubation ofthe A68 preparation, desirably with either Protein A or Protein G, andpreferably with both Protein A and Protein G, optimally which have firstbeen immobilized on agarose beads. Desirably, about 1 ml of a partiallypure A68 preparation is added to packed Protein A beads (generally about75 μl) and packed Protein G beads (generally about 75 μl). The samplepreferably is placed on a rotator, optimally for about 8 hours at 4° C.After incubation, desirably the beads are spun out of solution, e.g.,using a microcentrifuge at about 14,000×g for about 3 minutes. The A68supernatant preferably then is transferred to a new tube containingpacked Protein A and Protein G beads (generally about 75 μl of each) andallowed to incubate, optimally for up to 16 hours on a rotator at 4° C.Subsequently, the Protein A and G beads are pelleted, optionally using amicrocentrifuge at about 14,000×g for about 3 minutes. The A68supernatant is then stored (e.g., in 250 μl aliquots at −80° C.).

Alternately, immunoglobulin G desirably is removed by incubation of theprotein preparation with an immunoglobulin G removal method that issubstantially equivalent to use of both Protein A and Protein G, suchas, for instance, use of fixed bacteria or Pansorbin.

Total protein concentration of the purified A68 antigen preparationdesirably is determined by a Coomassie Blue protein assay, for instance,using Coomassie Plus Protein Assay Reagent (Pierce catalog #23236) andthe microassay as described by the manufacturer with knownconcentrations of BSA as a standard. Subsequently, reactivity of theantigen preparation with the A68 specific monoclonal antibody MC15desirably is determined by chemiluminescent indirect ELISA, and theactivity expressed as relative light units (rlu)/ng protein. Proteinconcentrations of subsequent A68 antigen preparations desirably can beestimated by comparing MC15 reactivity of the subsequent antigenpreparations against MC15 reactivity of the initial lot. This methodcircumvents problems of variability typically encountered when A68protein concentration is measured using a Coomassie Blue protein assay.

Determination of the immunoglobulin G content of the Protein A/G treatedA68 (or the partially purified A68 preparation) desirably can be done bychemiluminescent indirect ELISA using purified human IgG (Sigma, St.Louis, Mo.) as a standard, or by other appropriate means (i.e.,particularly means described in the Examples).

Using these methods of the invention, an A68 antigen preparation can beobtained that is substantially free of immunoglobulin G—i.e., which hasan amount of immunoglobulin G that preferably is equal to or less thanabout 0.05% (and even more preferably is equal to or less than about0.0015%) of the total protein of the preparation, and/or desirably whichhas less than about 500 pg of immunoglobulin G (and even more preferablyhas less than about 15 pg of immunoblobulin G) per μg of A68. When theantigen is used for Western blot analysis, the purified A68 preparationpreferably contains less than about 500 pg of IgG, and more desirablycontain less than about 15 pg IgG per amount of antigen loaded per gellane. It further is envisioned that substantially immunoglobulin G-freeantigen preparations also can be achieved using methods for “blocking”or “tying-up” any IgG present, or other appropriate means of removal ofthe IgG. Such methods include, but are not limited to: caprylic acidprecipitation of A68; adsorption using an anti-human IgG resin; and useof Pansorbin.

Assays Making Use Of The Alzheimer's Antigen Protein Preparation

This invention is directed inter alia to the detection of antibodies(autoantibodies) specific for Alzheimer's disease-associated antigen,which antigen is present in individuals with Alzheimer's disease andsubstantially absent from individuals who do not have Alzheimer'sdisease. The present invention provides specific and sensitive assaysfor diagnosis of Alzheimer's disease (i.e., for detecting the presenceof autoantibodies to the AD antigen). The methods of the inventionovercome the drawbacks of the prior art which require a diagnosis basedon a process of elimination of other disorders, and thus provide clarityto an assessment of treatment options.

Thus, this invention desirably is directed to detection of antibodies(i.e., autoantibodies) towards an Alzheimer's disease-associated antigenpresent in individuals with Alzheimer's disease and substantially absentfrom individuals who do not have Alzheimer's disease. This invention isalso directed to the detection of autoantibodies specific for tauproteins from human, as well as, other species (i.e., desirably amammalian species), such as bovine tau. Such tau protein antibodies arepresent in individuals who do not have AD, and are substantially absentfrom individuals with AD. The present invention provides a specific andsensitive assay for diagnosis of AD. Diagnosis is made based on therelative levels of Alzheimer's antibodies and MAPf autoantibodiespresent in body fluids, such as serum, plasma, and cerebrospinal fluid,such that individuals with substantial levels of Alzheimer's antibodies,and without substantial levels of autoantibodies to MAPf, are diagnosedas having AD.

This method accordingly provides for the use of Protein A/G treated A68(i.e., an A68 preparation that is substantially free of immunoglobulinG) as an antigen for detecting autoantibodies which are diagnostic forAlzheimer's disease. It is a novel and unexpected finding of theinvention that A68 purified according to the invention to besubstantially free of IgG, but not partially purified A68 preparations,can be employed in various methods (e.g., Western blot analysis,chemiluminescent sandwich ELISA assay, chemiluminescent indirect ELISAassay, direct ELISA assay, immunoprecipitation assays, and others) todetect autoantibodies specific for Alzheimer's disease. It will beapparent to one skilled in the art that these assays may be conducted inmany ways including direct and/or indirect ELISA, sandwich ELISA,Western blot analysis, etc. Furthermore, it will be apparent thatcompetition assays with any of the various Alzheimer's disease antigensand/or their precursors or related proteins, either alone or incombination, can aid in the detection of autoantibodies diagnostic forAlzheimer's disease.

In these assays according to the invention, desirably, antibodiesdirected against the Alzheimer disease antigen can be employed. Theseantibodies include monoclonal antibodies (e.g., as described in PCTInternational Application WO 96/20218) as well as serum autoantibodies.Certain preferred monoclonal antibodies are described in the Exampleswhich follow. However, one particularly preferred antibody is ALZ-50secreted by hybridoma No. HB9205, which was deposited under the BudapestTreaty on Sep. 17, 1986 with the American Type Culture Collection, 10801University Blvd., Manassas, Va. 20110-2209. ALZ-50 has become thestandard reagent for detecting the presence of Alzheimer's disease inthis field. (See, for example, Wood et al., Histochemical Journal, 21,No. 11, pp. 659-662 (1989); Itagaki et al., Annals of Neurology, 26, No.5, pp. 685-689 (1989); Beach et al., Brain Research, 501, No. 1, pp.171-175 (1989); Love et al., Journal of Neuropathology and ExperimentalNeurology, 47, No. 4, pp. 393-405 (1988); Nukina et al., NeuroscienceLetters, 87, No. 3, pp. 240-246 (1988); and Hyman et al., BrainResearch, 450, pp. 392-397 (1988).)

In the assay methods described herein, the sample used in the assay ofthe invention is preferably selected from the group consisting of braintissue, pre or post-mortem, cerebrospinal fluid, urine and blood. In apreferred embodiment, the sample comprises serum. The methods describedherein for use with serum are applicable to CSF and urine. The followingis another test procedure is believed to be suitable for detecting thepresence of autoantibodies to Alzheimer antigen in the blood or otherbody fluids of a person having Alzheimer's disease. The procedure issimilar to the procedure used in the detection of HTLV-III as disclosedin “Immunoassay for the Detection and Quantitation of Infectious HumanRetrovirus, Lymphadenopathy-Associated Virus (LAV)”, by J. S. McDougalet al., Journal of Immunological Methods, 76, pp. 171-183 (1985).Preferably from about 0.1 μl to about 100 μl of serum is utilized, morepreferably 0.25 μl to 10 μl of serum is utilized.

Thus, in terms of a Western analysis for detection of autoantibodiesthat are present in Alzheimer's disease, the present invention providessuch a method that optimally comprises:

(a) obtaining an A68 purified protein preparation according to theinvention, and a sample being tested for the presence of theautoantibodies;

(b) electrophoresing the protein preparation on a gel;

(c) transferring the electrophoresed protein preparation to a membrane(e.g., nitrocellulose);

(d) contacting the membrane with a sample being tested for the presenceof the autoantibodies such that an antigen-autoantibody complex canform; and

(e) detecting the autoantibodies by the formation of the complex.

Similarly, in the instant invention, a method is provided fordetermining the presence of autoantibodies specific to Alzheimer'sdisease in a sample, thereby diagnosing Alzheimer's disease. The methodoptionally comprises contacting a sample from an individual suspected ofhaving Alzheimer's disease with a purified A68 antigen preparationaccording to the invention. In terms of a sandwich ELISA assay (e.g.,described in the Examples), this contacting optimally is done after thesample has been allowed to bind to Protein A/G (preferably which hasbeen immobilized on beads, plates, nitrocellulose, fixed bacteria,Pansorbin, and the like). This contacting optionally is done such thatthe autoantibody is free in solution, and immobilized subsequent tocontacting the Ag. Subsequently (e.g., following washing), the mixturedesirably is contacted with an antibody specific for an antigenicdeterminant on the Alzheimer's antigen (e.g., ALZ-50 or monoclonal TG5specific for A68) and capable of binding so as to produce a complex.According to the invention, the “antibody” can be a portion of anantibody (e.g., a Fab fragment, etc.). The resulting complex thenoptionally can be separated and recovered from the sample, butpreferably, is detected by an appropriate means, e.g. chemiluminescentmeans, etc.

Optionally, A68 is labeled, as with biotin or radioactive markers bystandard protocols, and the complex desirably is measured by detectionof that label. This is accomplished, for instance, with reagents such asstreptavidin conjugated to horseradish peroxidase in the case ofbiotin-labeled A68, or through capture of the complex and detection ofradioactivity in that complex. Labeling means and means of detectinglabels are well known to those skilled in the art.

Accordingly, the invention further provides a method for detectingautoantibodies that are present in Alzheimer's disease comprising:

(a) obtaining a purified A68 protein preparation according to theinvention;

(b) contacting the protein preparation with a sample being tested forthe presence of the autoantibodies such that an antigen-autoantibodycomplex can form; and

(c) detecting the autoantibodies by the formation of the complex.

As previously described, the method desirably can be carried out wherethe presence of the autoantibodies is determined by the presence of thecomplex (i.e., a qualitative test). Optionally, the method can becarried out where the amount of the complex is measured, and the amountof the autoantibodies is determined by the amount of the complex (i.e.,a quantitative test).

Additionally, the method optionally can comprise the further step ofcontacting the complex with an antibody that is immunologically reactivewith an antigenic determinant found on either the autoantibody or theprotein preparation such that an antigen-antibody orantibody-autoantibody complex is formed.

The antibodies employed in the methods of the present inventionoptimally can be made detectable by attaching an identifiable labelthereto. The antibody preferably is made detectable by attaching to itan enzyme conjugated to an appropriate substrate which, in turn,catalyzes a detectable reaction. The enzyme may be horseradishperoxidase, beta-galactosidase or alkaline phosphatase. Other means ofdetection of the antibody include attaching a fluorescent,chemiluminescent, or radiolabel thereto. Alternatively, the antibody maybe detected by use of another antibody directed to it, the otherantibody being labeled or having an enzyme substrate bound to it. Thepresence of the detectable antibody (e.g., as an indicator of thecomplex) may be readily detected using well-known techniques. Thus, ifthe detectable antibody is linked to an enzyme and introduced to anappropriate substrate, the optical density of the detectable boundantibody is determined using a quantum spectrophotometer. If thedetectable antibody is fluorescently labeled, the fluorescent emissionmay be measured or detected using a fluorometer technique. In a similarmanner, if the detectable antibody is radioactively labeled, the boundantibody may be detected using radioactivity detection techniques. Bycomparing the results obtained using the above-described methods on thetest sample with those obtained using the methods on a control sample,the presence of the purified A68 protein preparation/autoantibodycomplex specific to Alzheimer's disease may be determined. The elevatedamount of purified A68 protein preparation/autoantibody specific toAlzheimer's disease is thereby detected and may optionally bequantitated.

The methods for qualitatively or quantitatively determining theAlzheimer's disease antigen/autoantibody complex may be used in thediagnosis of Alzheimer's disease. Utilization of the methods of thepresent invention is advantageous over prior art methods because thepresent invention provides simple, sensitive, very specific methods fordetecting Alzheimer's antigen/autoantibody complex. The Alzheimer'santigen is well-suited for sandwich immunoassay complex formation sinceit is present in aggregate form and, hence, is multiepitopic. This is incontrast to cross-reactive proteins, which are soluble and usuallycontain one epitope per protein.

Variations of these and other standard methods for detection ofautoantibodies and autoantigens would be evident to one skilled in theart, and are contemplated by the invention.

Method of Making Alzheimer Disease Antigens for Detecting Autoantibodies

The invention also desirably provides a method of increasing the abilityof an Alzheimer's disease antigen to detect autoantibodies that arepresent in Alzheimer's disease, wherein the antigen is tau isolated fromvarious species including human, or is recombinant human tau. Theresulting antigen preparation optionally can be employed instead of (orin addition to) the substantially pure A68 preparation of the invention.

One such method comprises phosphorylating the antigen. Preferably thephosphorylation is done using a cell extract prepared from a centralnervous system (CNS) cell line, e.g. neuroblastoma cells (especially MSNneuroblastoma cells), optionally which has been treated with aphosphatase inhibitor, such as okadaic acid. Also, desirably thephosphorylation is done using a purified or partially purified kinasewhich has been associated in the literature with tau phosphorylation.Such kinases which can be used in the context of the invention include,but are not limited to, PKA, GSK, cdc2, cdc25, casein kinase I and II,MAP kinase, and PHF kinase.

The invention further provides a method of increasing the ability of anAlzheimer's disease antigen to detect autoantibodies that are present inAlzheimer's disease, preferably wherein the antigen is tau isolated fromvarious species including human, or is recombinant human tau, orphosphorylated recombinant human tau (Ptau) or phosphorylated isolatedtau, and the method comprises optionally treating the antigen withhypericin.

Similarly, the invention desirably provides a method of increasing theability of an Alzheimer's disease antigen to detect autoantibodies thatare present in Alzheimer's disease, wherein the antigen is tau isolatedfrom various species including human, or is recombinant human tau (rht),or is phosphorylated recombinant human tau (phospho-rht) orphosphorylated isolated tau, and the method comprises treating theantigen with free fatty acids. Preferably the fatty acids areunsaturated fatty acids, particularly oleic or linoleic acids, and mostpreferably arachidonic acid.

Moreover, the invention also provides a method of increasing the abilityof an Alzheimer's disease antigen to detect autoantibodies that arepresent in Alzheimer's disease, wherein the antigen is tau isolated fromvarious species including human, or is recombinant human tau, orphosphorylated is recombinant tau (phospho-rht) or phosphorylatedisolated tau, and the method optionally comprises treating the antigenwith advanced glycation endproducts, especially where the advancedglycation endproduct is the lipid peroxidation product4-hydroxy-2-nonenal (HNE).

Anti-Idiotypic Antibodies

The invention further provides a means of obtaining so-called“anti-idiotypic antibodies”, which are antibodies that recognize aminoacid differences in, and hence are specifically directed to, particularimmunoglobulins. In particular, the invention preferably provides meansof identifying anti-idiotypic antibodies to A68 Alzheimer's diseaseantigen-reactive immunoglobulins, starting from either monoclonalantibodies to A68 or human serum autoantibodies to A68. Theseanti-idiotypic antibodies desirably are employed in the methods of theinvention for assaying for Alzheimer's disease.

Accordingly, the invention desirably provides an antibody (e.g.,especially a monoclonal antibody) that is immunologically reactive withan antibody (e.g., especially a monoclonal antibody, or human serumautoantibody) directed against A68 antigen. Such an anti-idiotypicantibody, especially an antibody that is immunologically reactive with amonoclonal antibody or human serum autoantibody directed against A68antigen, desirably is obtained by:

(a) obtaining sera from individuals having high titers of anti-A68autoantibodies, combining to create a pool, and isolating antibodiesfrom said pool,

-   -   or, obtaining isolated monoclonal antibodies to A68 antigen;

(b) immunizing mice with the isolated antibodies;

(c) obtaining serum from the mice (i.e., after sufficient time and undersufficient conditions for antibodies to be produced); and

(d) testing the serum to identify mice having high levels of antibodiesthat are immunologically reactive with an antibody (e.g., a monoclonalantibody or human serum autoantibody) directed against A68 antigen.

The method optionally can be carried out comprising the further steps:

(a) obtaining the spleens of the mice having high levels of antibodiesthat are immunologically reactive with a monoclonal antibody or humanserum autoantibody directed against A68 antigen;

(b) fusing the spleens with myeloma cells and plating onto tissueculture plates;

(c) selecting for fused cells by HAT resistance; and

(d) testing said fused cells for production of antibodies that areimmunologically reactive with an antibody (e.g., a monoclonal antibodyor human serum autoantibody) directed against A68 antigen. Optimally themethod further comprises testing the fused cells for production ofantibodies that are not immunologically reactive with antibodies notdirected against A68 antigen. Suitable variations of these methods willbe apparent to those skilled in the art.

Bovine Tau (MAPf) Preparation and Assays

This invention further desirably provides for the use of a bovinemicrotubule-associated protein preparation (i.e., MAPf) in conjunctionwith A68, for instance, in Western blot analysis of sera. Bovine MAPfcontains, among other things, 70% MAPs 1 & 2, 20% other MAPs, and 10%Tau MAP isoforms. The current invention, as described herein, makes useof the six Tau MAP isoforms that migrate in the 40-65 kD range on a 10%SDS polyacrylamide gel. The methods for electrophoresis, Westerntransfer, sera incubation, and detection of bound autoantibody are wellknown to those skilled in the art.

Thus, desirably according to the invention, an individual can employ forelectrophoresis alternating lanes of A68 and MAPf. MAPf preferably isused at the concentration of 1.5 ug total protein/lane. Followingelectrophoresis, the protein is transferred to an appropriate support,e.g., nitrocellulose, or other membrane. Desirably, patient sera isincubated with the strips of nitrocellulose containing purified A68protein preparation and strips of nitrocellulose containing MAPf, andbound autoantibodies are then visualized as previously described. Underthese conditions, bound autoantibodies to purified A68 proteinpreparation and the tau isoforms in MAPf desirably are obtained.

Accordingly, the present invention also desirably provides a method fordetecting autoantibodies that are present in Alzheimer's diseasecomprising the steps of:

(a) obtaining a purified A68 protein preparation as previouslydescribed, a bovine microtubule associated protein preparation, and asample being tested for the presence of autoantibodies;

(b) electrophoresing the A68 protein preparation and the bovinemicrotubule associated protein preparation on separate lanes on a gel;

(c) transferring the electrophoresed A68 protein preparation and thebovine microtubule associated protein preparation to a membrane;

(d) contacting the membrane with a sample being tested for the presenceof said autoantibodies such that an autoantibody complex can form withantigen present in the A68 protein preparation and/or with antigenpresent in the bovine microtubule associated protein preparation; and

(e) detecting the autoantibodies by the formation of the complex(es).

Two methods of analysis preferably are used to assign a diagnosis toeach serum tested. In the first method, the total optical density(OD)×mm signal from purified A68 protein preparation is divided by thetotal OD×mm signal from the tau isoforms. In general, the sample isassigned the diagnosis of AD, or non-AD on the basis of this ratio.Optical density can be calculated, for instance, as described in Example11.

The second method optimally takes into account not only optical densitymeasurements, but also the number of MAPf tau isoforms identified by agiven serum. In this method of analysis, if a bovine MAPf tau signal ispresent in conjunction with purified A68 protein preparation, desirablythe sample is assigned a diagnosis of AD if the tau signal contains lessthan three isoforms, and a diagnosis of non-AD if the sample identifies3 or more isoforms of tau. In addition, preferably the sample isclassified as non-AD if it lacks purified A68 protein preparationsignal, regardless of the number of tau bands. Quantification of theMAPf tau signal in this instance takes on the formula: (Sum OD×(n−2)).For this formula, “Sum OD” is the sum of OD×mm measurements of all tauisoforms and “n” is the total number of bands present of the six tauMAPf isoforms. Thus, samples that give a purified A68 proteinpreparation signal and lack a substantial tau signal by this method aretermed AD, and all other combinations (A68 signal+tau signal, tau signalalone, or absence of both signals) are diagnosed as non-AD.

Preferably the use of tau isoforms is not limited to use of bovine tauisoforms found in MAPf. Other forms of tau protein desirably are used,including but not limited to, tau purified from brain, and recombinanttau, either as a single molecule or as a mixture of tau isoforms.Additionally, the invention is not limited to tau from a bovine species.Purified tau or MAP from brains or cultured cells of other species maybe used such as human, rodent, or other mammalian sources, as well aspreparations from avian and reptiles.

Similarly, autoantibodies reactive with purified A68 protein preparationand bovine tau may also be detected in an indirect ELISA assay whereinthe antigen is immobilized in a microtiter plate in which the bottom ofeach well is nitrocellulose. This support allows the antigen to bedisplayed in a manner which more closely resembles the Western blot thandoes a polystyrene support. Millipore MHAB plates are prewet with BBSfor 1 minute, then the buffer is drawn through the filter under vacuum.Antigen is applied to the wells in BBS at 0.01 to 10 μl per well (0.3 to300 ng), and allowed to bind for 3 hours at 24° C. In these experiments,the antigen may be purified A68 protein preparation, bovine tau (MAPf),or purified A68 protein preparation analogues such as phosphorylatedrht. The antigen solution is drawn through the filter under vacuum, andthe filters are blocked with 5% non-fat dry milk in BBS for 1.5 hr at24° C. Subsequently, all incubation solutions are removed by platewasher (Nunc) and washed with 0.1% tween 20 in tbs (defined earlier)rather than by drawing through the membrane under vacuum. 1% serum isadded to wells in 100 μl of 1% non-fat dry milk, 5% normal goat serum,BBS, and incubated for 16 hr at 4° C. Bound human Ig is detected byaddition of HRP-conjugated goat anti-human Ig in 1% casein/tbs for 2 hrat 24° C. followed by addition of 90 μl LumiGlo (Kirkegaard and Perry)chemiluminescent substrate. Chemiluminescence is measured as describedin Example 5.

EXAMPLES

The invention is illustrated further by the following examples which arenot to be construed as limiting the invention in scope or spirit to thespecific compounds or procedures described in them, or as in any otherway limiting the invention's scope.

Example 1 Isolation of a Partially Purified A68 Antigen Preparation

This Example describes the isolation of a partially purified A68 antigenpreparation.

A68 antigen was isolated from frozen human brain samples (typically thecerebral cortex from an Alzheimer's disease patient), by homogenizationin 5 volumes of an aqueous buffer such as tris buffered saline (TBS),containing standard protease and phosphatase inhibitors. The homogenatewas fractionated by centrifugation at 27,000×g for 60 minutes at 4° C.,and the supernatant was collected and passed over an MC1 affinity columniteratively for 16 hours at 4° C. The MC1 column was prepared bycoupling a purified mouse monoclonal antibody which reacts specificallywith A68 (MC1, described in PCT International Application WO 96/20218,and deposited in terms of its source, secreting hybridoma ATCC No.11736, with the American Type Culture Collection, Rockville, Md. on Oct.26, 1994) to Affigel-10 (Biorad Laboratories) according tomanufacturer's instructions. A68 is specifically removed from thesupernatant by the MC1 column matrix. Following extensive washing withTBS, A68 was eluted from the MC1 column using 3 M KSCN. It subsequentlywas dialyzed against TBS and stored at −80° C. Other means of isolatinga partially purified A68 antigen preparation (which in some instancesare similar, if not identical, to that above) are described in PCTInternational Application WO 96/20218.

The obtained preparation is highly enriched in A68, but also containsamounts of other proteins. The quantity of human Ig in A68 wasascertained through indirect ELISA wherein A68 is coated onto an ELISAplate and probed with horseradish peroxidase (HRP)-conjugated goatantibodies reactive specifically with human Ig. Standard amounts ofpurified human Ig are coated in other wells and used as standards. BoundHRP-conjugated Abs are quantitated using chemiluminescent HRPsubstrates, and the quantities between the standard human Ig and the A68preparations are compared. Surprisingly, the A68-enriched preparationwas found to contain endogenous human immunoglobulins comprisingapproximately 1-5% of the total protein. This is surprising since suchimmunoglobulins would be expected to be removed during affinitychromatography. As confirmed by the Examples below, this unexpectedlyhigh level of immunoglobulin interferes with the ability of thepreparation to detect serum autoantibodies to A68 by either Westernblotting or ELISA.

Example 2 Protein A/G Treatment of A68 Preparations

This Example describes the further purification with use of protein A/Gof an A68 antigen preparation obtained, for instance, as described inExample 1. Unless otherwise specified, all chemicals for this study, andthose in the following Examples, were purchased from Sigma (St. Louis,Mo.).

To remove the contaminating Ig's prior to analysis of serumautoantibodies, the A68 preparation was incubated with both Protein Aand Protein G immobilized on agarose beads (Immunopure ImmobilizedProtein A, Immunopure Immobilized Protein G; Pierce, Rockford, Ill.).Briefly, 1 ml of A68 was added to 75 μl of packed Protein A beads and 75μl of packed Protein G beads. The sample was placed on a rotator for 8hours at 4° C. After incubation, the beads were spun out of solution ina microcentrifuge at 14,000×g for 3 minutes. The A68 supernatant wasthen transferred to a new tube containing 75 μl each of packed Protein Aand Protein G beads and allowed to incubate for an additional 16 hourson a rotator at 4° C. Subsequently, the Protein A and G beads werepelleted using a microcentrifuge at 14,000×g for 3 minutes, and the A68supernatant was then stored in 250 μl aliquots at −80° C. Determinationof IgG content of the Protein A/G treated A68 was done bychemiluminescent indirect ELISA using purified human IgG (Sigma, St.Louis, Mo.) as a standard as described in Example 1. The preparation wasfound to be essentially free of endogenous IgGs, having an amount of IgGequal to or less than 0.05% of the total protein of the sample.

Example 3 Western Blot Analysis

This Example describes Western blot analysis of an A68 antigenpreparation that has been purified to be substantially free ofimmunoglobulin by treatment with Protein A/G, as described in thepreceding Example.

Gel electrophoresis was performed using the method of Laemmli (Nature,227, pp. 680-685 (1970)), employing 10% SDS-polyacrylamide minigels ofabout 1.5 mm thickness. Protein A/G treated A68 was loaded at about100-1000 ng total protein/lane in sample buffer.

Western transfer was performed according to the method of Towbin et al.(Proc. Natl. Acad. Sci. USA, 76, pp. 4350-4354, (1979)) using either0.45 μm or 0.2 μm nitrocellulose (Micron Separations Inc. Westboro,Mass.). Following transfer, the nitrocellulose blots were removed andput into blocking buffer consisting of 5% nonfat dry milk in boratebuffered saline (BBS, 75 mM NaCl, 100 nM H₃BO₃, 25 mM B₄Na₂O₇:10H₂O) forabout 2 hours at room temperature. The nitrocellulose was then cut toseparate the individual lanes of protein. The strips of nitrocellulosewere incubated on a rocker with patient serum at dilutions of 1:100 to1:1200, for about 16 hours at 4° C. in 1% nonfat dry milk in BBS+5%normal goat serum. Subsequently, the strips were washed twice, 5 minuteseach wash, with BBS+0.05% Tween-20, followed by a final 45 minute washwith BBS+Tween-20. Washes were performed at ambient temperature on ashaker.

Goat-anti-human IgG-HRP antibody (Southern Biotechnology Associates,Birmingham, Ala., Cat #2040-05) was then added to the strips at about0.1 μg/ml in 1% nonfat dry milk in BBS and the strips were incubated forabout 1.5 hours on a shaker at ambient temperature. The nitrocellulosestrips were then washed 4 times, 5 minutes each, with BBS+0.05%Tween-20. The strips were then soaked for 5 minutes in ECL (LumiGLO,Kirkegaard and Perry, Gaithersburg, Mass., Cat. #50-59-00). The excessLumiGLO was allowed to drain from the strips, which were then placed ina plastic page holder. The strips were then overlaid with preflashedX-ray film (Hyperfilm, Amersham, Arlington Heights, Ill.) for 1-30 min.Preflash of the X-ray film was carried out using the Amersham Sensitizeunit as prescribed by the manufacturer. The X-ray film was thendeveloped by standard methods to visualize the A68 signal.

In terms of positive and negative controls for the Western analysis,sera were run at several dilutions ranging from 1:100 to 1:1200. Thesera were incubated with nitrocellulose strips (i.e., corresponding togel lanes) that contained protein A/G-treated A68 in sample buffer, orsample buffer alone. Additionally, a lane of protein A/G A68 wasprocessed as described, with the exception that human sera was left outin the first incubation step (i.e., the lane was probed withgoat-antihuman IgG-HRP antibody only). Both of these measures serve asnegative controls. As a positive control, one lane of A68 was probedwith a monoclonal antibody to A68, such as Alz50 or TG5 (i.e., PCTInternational Application WO 96/20218, and TG5 is further discussedbelow). Sera were scored as positive if they exhibited positive stainingof A68 bands in the 760-70 kD range on the Western blot, but otherwise,were negative in the lanes that contained sample buffer only. Sera werescored as negative if they lacked visible bands in the 60-70 kD range.

Results of these studies are set out in FIGS. 1 and 2. As can be seenfrom FIG. 1, autoantibodies to A68 were present in individuals withAlzheimer's disease (lanes 2 and 4), while normal controls lackedreactivity with the A68 protein (lanes 3 and 5).

Next, the necessity of protein A/G treatment of the A68 preparation forthe detection of serum autoantibodies in Alzheimer's disease by Westernblotting was ascertained, and is presented in FIG. 2. Two lots ofuntreated A68 prepared as described in Example 1 (Lot A, lanes 1-3 andLot B, lanes 4-6) and one lot of Protein A/G treated A68 as described inExample 2 (lanes 7-9) were run on a 10% SDS polyacrylamide gel andtransferred to nitrocellulose. Total protein loaded was approximately175 ng/lane. The strips were probed with sera from two Alzheimer'sdisease patients (Patient 1, lanes 2, 5, 8 or Patient 2, lanes 3, 6, 9)followed by goat anti-human IgG-HRP, or the strips were probed for IgGendogenous to the A68 preparations using the goat anti-human IgG HRPantibody only (lanes 1, 4, 7). Blotting of untreated A68 shows thepresence of a 55 kD band representing the heavy chain of IgG (indicatedby the arrow in FIG. 2). Furthermore, the untreated A68 reacted weaklyor not at all with autoantibodies in human sera. Protein A/G treated A68lacked endogenous IgG reactivity but was recognized strongly by serumautoantibodies to A68 as evidenced by the prominent A68 bands (indicatedby the arrowhead in FIG. 2). Protein A/G treatment not only eliminatedendogenous IgGs but also enriched for A68 resulting in enhancedautoantibody reactivity.

This Example confirms that Western blot assays using an A68 antigenpreparation that has been purified to be substantially free ofimmunoglobulin by treatment with Protein A/G (as described in Example2), but not A68 antigen preparation that have not been subjected to thisadditional level of purification, can be employed to detect Alzheimer'sdisease autoantibodies.

Example 4 Chemiluminescent Sandwich ELISA Assay

This example describes a chemiluminescent sandwich ELISA assay for thedetection of autoantibodies to A68.

For these studies, Dynex microtiter plates type Microlite 1 were coatedwith 5 μg/ml Pierce Protein A/G for 3 hours at 24° C. in 25 mM NaPO₄, pH7.2, 125 mM NaCl, 2 mM EDTA, 2 mM NaN₃, (coat buffer) and weresubsequently blocked with a diluent (Casein/TBS) consisting of 1%casein, 10 mM Tris-Cl, pH 7.4, 140 mM NaCl, and 1 mM NaN₃ for 1 hour at24° C. Human serum or plasma, collected and prepared according tostandard practices, was incubated in the wells as a 1% solution inCasein/TBS, and allowed to bind for about 3 hours at 24° C. Protein A/Gpurified A68 was then added at 70 ng/ml in Casein/TBS, and incubated inthe wells for 90 hours at 4° C.

TG5, a mouse monoclonal IgG which is highly selective for A68 (describedin PCT International Application WO 96/20218, and deposited in terms ofits source, secreting hybridoma ATCC No. HB 11746, with the AmericanType Culture Collection, Rockville, Md. on Oct. 26, 1994), was processedto produce a F(ab′)₂ fragment conjugated with horseradish peroxidase(IRP, see below). This conjugate was incubated in the wells of the sameplates that were pretreated with human serum or plasma at 0.25 μg/ml for3 hours at 24° C. in Casein/TBS, to react with the A68 that had beencaptured by autoantibodies. Bound TG5-HRP was then detected using aluminol-based chemiluminescent substrate (i.e., Lumiglo, Kirkegaard andPerry). Luminescence was quantitated with a Labsystems luminometer athigh gain using a 5 sec/well read time. All incubations were 100 μlexcept for the blocking step, which was 288 μl, and the luminol, whichwas 88 μl. In between each step, the microtiter wells were washed 5times with a solution of 0.1% Tween 20 in 10 mM Tris-Cl, pH 7.4, 140 mMNaCl, 1 mM NaN₃ (TBS) to remove unbound materials.

TG5 was prepared for these studies as follows. TG5 was purified fromtissue culture supernatants on immobilized Protein A. It was dialyzedinto 50 mM NaPO₄, pH 8.1, at a concentration of greater than 5 mg/ml. Itwas digested to a F(ab′)₂ with immobilized ficin according tomanufacturer's instructions (Pierce). The F(ab′)₂ fragment was removedfrom the Fc fragment and residual intact IgG by passing the digest overa Protein A column. To generate a Fab′, TG5 was dialyzed against 0.1 MNaPO₄, pH 6.0, 5 mM EDTA and reduced with 6 mg/ml mercapto-ethylaminefor 90 minutes at 37° C. The buffer was changed to 0.1 M NaPO₄, pH 7.0,5 mM EDTA by desalting on a Sephadex G25 column, and the Fab′ containingfractions were pooled and concentrated to greater than or equal to 1mg/ml. Pierce maleimide-HRP, at a mass ratio of 1:2, TG5:HRP, was addedand allowed to react for 1 hour at 24° C., then an additional 16 hoursat 4° C. The resulting conjugate was used without further processing.

Three serum samples obtained from patients which were clinicallydiagnosed as having Alzheimer's disease according to NINCDS-ADRDAcriteria were analyzed using the above protocol. Results of thesestudies are shown in Table 1.

TABLE 1 Autoantibody Detection in Clinical Specimens Relative LightUnits, blank Sample # Clinical Diagnosis subtracted DT AD 9.01  836 AD2.04 5114 AD 1.21For each of the three samples, autoantibodies were detected as can beseen from the signal expressed in relative light units in Table 1.

This Example confirms that a chemiluminescent sandwich ELISA assay usingan A68 antigen preparation can be employed to detect Alzheimer's diseaseautoantibodies.

Example 5 Chemiluminescent Indirect ELISA

This example describes a chemiluminescent indirect ELISA assay for thedetection of autoantibodies to A68.

For these studies, Protein A/G-treated A68 was added to bicarbonatecoating buffer (35 mM NaHCO₃, 15 mM Na₂CO₃, pH 9.65, 0.2 μm sterilefiltered) at a concentration of 1 μg/ml, and 100 ul/well was added to aMICROLITE 2 microtiter plate (Dynex Technologies, Chantilly, Va.). TheA68 was allowed to adsorb to the wells for 2 hours at 25° C., or 16hours at 4° C. For determination of background, corresponding wells werecoated with bicarbonate buffer only. The wells are then washed 3 timeswith wash buffer (10 mM Tris(hydroxymethyl)-aminomethane, 150 mM NaCl,0.1% Tween-20, pH 7.4) using a 12 well washer (Immuno Wash 12, Nunc,Denmark). Subsequently, the wells were blocked for 2 hours at 25° C.with 300 μl/well of 1% Casein/TBS (1% casein (sodium salt), 25 mMTris(hydroxymethyl)-aminomethane, 145 mM NaCl, 0.01% thimerisal, pH 7.5,sonicated for 6 hours at 10 watts with a Vibro Cell Sonicator at 25° C.(Sonics Materials, Danbury, Conn.), and then filtered through a 0.45 μmSFCA membrane). The wells were then washed 3 times with wash buffer.

Serum diluted 1:500-1:1000 in 1% Casein/TBS with 5% normal goat serumwas added to the wells (100 μl/well) and incubated at 25° C. for 2hours. The wells were then washed 8 times with wash buffer.Subsequently, 100 μl of 0.1 μg/ml goat antihuman IgG-HRP (SouthernBiotechnology Associates, Birmingham, Ala.) in 1% Casein/TBS was addedto the wells and allowed to incubate for 1.5 hours at 25° C. Followingthis incubation, the wells were washed an additional 8 times with washbuffer. 88 μl of ECL (LumiGLO, Kirkegaard and Perry, Gaithersburg,Mass.) was added to the wells and the wells were counted for 2 secondseach at 21° C., integral mode, using a Lumionskan RS luminometer(Labsystems, Finland). Unless otherwise specified, all chemicals werepurchased from Sigma (St. Louis, Mo.).

To determine the absolute value for a serum sample the relative lightunit (RLU) signal from sera in the bicarbonate buffer-only wells wassubtracted from the RLU signal from sera in the A68-coated wells.Plate-to-plate and day-to-day variations were corrected for by using anA68 standard curve ranging from 25 ng/well with doubling dilutions to0.1 ng/well. Detection of the A68 in the standard curve was accomplishedby using the A68-specific monoclonal antibody MC15 (1 μg/ml, describedin PCT International Application WO 96/20218, and deposited in terms ofits source, secreting hybridoma ATCC No. HB 11739, with the AmericanType Culture Collection, Rockville, Md. on Oct. 26, 1994) and asecondary goat anti-mouse IgM-HRP (0.1 μg/ml, Southern BiotechnologyAssociates), both diluted in 1% Casein/TBS.

Results of this assay are depicted in Table 2.

TABLE 2 Indirect ELISA for autoantibody detection Relative Light Units(background Sample # Clinical Diagnosis substracted) 5186 AD 898.8 8131AD 432.9 5178 AD 267.1 5188 AD 150.5 6010 AD 95.8As can be seen from Table 2, autoantibodies were detected by theindirect ELISA in all five AD patients, as confirmed by the signalexpressed as relative light units.

This Example confirms that a chemiluminescent indirect ELISA assay usingan A68 antigen preparation purified according to the invention can beemployed to detect Alzheimer's disease autoantibodies.

Example 6 Detection of Autoantibodies in AD

This example describes the production and use of anti-idiotypicantibodies for the detection of autoantibodies in Alzheimer's disease.

Monoclonal antibodies to A68 and/or human serum autoantibodies to A68are used to produce monoclonal anti-idiotypic antibodies. Theanti-idiotypic antibodies are then used as the antigen in an indirectELISA to screen for anti-A68 autoantibodies in serum from individualssuspected of having Alzheimer's disease. Such use of anti-idiotypicantibodies forms the basis for a serum test for Alzheimer's disease.

Human sera from individuals with Alzheimer's disease are screened byWestern blotting against A68. Sera from individuals that are shown tohave high titers of anti-A68 autoantibodies are pooled. Human antibodiesare isolated from the pooled serum by batch incubation with Protein A/Gimmobilized on agarose beads. Alternatively, immunoaffinity columnsusing A68 can be used to enrich for A68 autoantibodies which aresubsequently eluted off of the column. The eluted antibodies are thencaptured as above, using Protein A/G beads. Typically, 1 ml of ProteinA/G binds 6-8 mg of IgG. For isolation of human IgG from sera, the serais diluted 1:1 with 10 mM Tris, pH 7.5 and incubated for 2 hours at 4°C. with 1 ml of Protein A/G beads. For immunoaffinity purified A68autoantibodies, or monoclonal antibodies in tissue culture soup, theProtein A/G beads are added directly to the antibody solution at aconcentration that is proportional to IgG content (i.e., about 1 ml ofProtein A/G beads per 6-8 mg of IgG).

Anti-idiotypic monoclonal antibodies are produced using standard methods(see Harlow, E. and Lane, D., Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory Press, (1988)). Briefly, Balb/c mice areimmunized by intraperitoneal injections consisting of 10-15 μl ofProtein A/G beads loaded with anti-A68 autoantibodies from sera asdescribed above (A/G A68 antibody/bead) in Freund's complete adjuvant.Subsequent boosts occur on days 14 and 21 with Protein A/G beads loadedwith anti-A68 autoantibodies (A/G-A68-Ab-beads) in Freund's incompleteadjuvant. On day 31, the mice are tail bled and the serum is tested forthe presence of anti-idiotypic antibodies by competition ELISA using A68(described below). Spleens of mice displaying high titers ofanti-idiotypic antibodies are fused with SP2/O-Ag8 myeloma cells andplated into 96 well tissue culture plates. Fused cells are selected forby HAT resistance and the resulting clonal populations are screened byindirect ELISA for the presence of anti-idiotypic antibodies to A68autoantibodies. Clonal populations that test positive for anti-idiotypicantibodies are further subcloned by standard techniques to producemonoclonal hybridoma populations.

Initial and subsequent clones for anti-idiotypic antibodies to A68autoantibodies are identified using an indirect ELISA. The followingprotocol is used to identify anti-idiotypic antibodies raised againstautoantibodies to A68: 96 well ELISA plates are coated with 100 μl/wellof 1 μg/ml goat anti-mouse IgG Fc specific antibodies in bicarbonatebuffer (35 mM NaHCO₃, 15 mM Na₂CO₃, pH 9.65, 0.2 μm sterile filtered)for 2 hours at 25° C. The wells are washed 3 times with wash buffer (10mM Tris(hydroxymethyl)-aminomethane, 150 mM NaCl, 0.1% Tween-20, pH 7.4)and subsequently blocked for 2 hours at 25° C. with 300 μl/well of 1%Casein/TBS.

The wells are washed 3 times with wash buffer. Tissue culture media fromthe hybridoma wells to be tested is then added to the microtiter plateat a dilution of 1:100 in 1% Casein/TBS and allowed to incubate for 2hours at 25° C. The wells are washed 3 times with wash buffer and thenpooled human sera from Alzheimer's disease patients that were used asthe original immunogen is added to the wells at a dilution of 1:100 in1% Casein/TBS. After a 2 hour incubation at 25° C., the wells are washed8 times with a wash buffer. Subsequently, 100 μl of 0.1 μg/ml goatanti-human IgG-HRP (Southern Biotechnology Associates, Birmingham, Ala.)in 1% Casein/TBS is added to the wells and allowed to incubate for 1.5hours at 25° C. Following this incubation, the wells are washed anadditional 8 times with wash buffer. 88 μL of ECL (LumiGLO, Kirkegaardand Perry, Gaithersburg, Mass.) is added to the wells and the relativelight units (RLU) are recorded by counting the individual wells for 1sec each at 21° C., integral mode, using a Lumionskan RS luminometer(Labsystems, Finland).

A duplicate plate is set up in parallel and treated the same way exceptin this case the sera is derived from normal individuals that lackautoantibodies to A68. Positive clones containing only anti-idiotypicantibodies of interest will react with sera containing autoantibodies toA68, but not to sera from normal individuals lacking theseautoantibodies. To screen clones that have been produced usingmonoclonal antibodies as the initial antigen, essentially the samestrategy is utilized with the exception that a direct ELISA is used. Inthis instance, the monoclonal antibody that was used as the immunogen ismodified to a F(ab′)₂ fragment and is conjugated to HRP. Thismethodology is necessary so that the monoclonal antibody used to detectthe anti-idiotypic antibody is not captured by the anti-mouse Fcantibody on the plate.

Further characterization of the anti-idiotypic monoclonal antibodies isaccomplished by use of a competition assay with A68. These assays areperformed by adding the anti-idiotypic antibody to the plate asdescribed above. Then, detection antibodies (either human serumantibodies or monoclonal F(ab′)₂ HRP conjugated antibodies) are addedtogether with various concentrations of A68. A68 will compete with theanti-idiotypic antibodies for the antigen recognition site on thedetection antibody resulting in a diminution of signal.

It should be noted that there are several variations of the above assaythat also could be used to screen for anti-idiotypic antibodies. Theseassays are obvious to those skilled in the art.

Example 7 Phosphorylated Recombinant Tau and Treatment with Hypericin

This Example describes the means by which recombinant human tau (rht)can be phosphorylated and optionally treated with hypericin to increaseits reactivity with Alzheimer's disease autoantibodies.

For these studies, a MSN extract was prepared by culturing MSN cells(Reynolds et al., J. Natl. Cancer Inst., 76: pp. 375-387, 1986) in T225flasks in RPMI 1640 (supplemented with 15% fetal bovine serum, 100 U/mlpenicillin, 100 ug/ml streptomycin) in a humidified incubator with a 5%CO₂ atmosphere. The cells were collected when 80% confluent by scrapingand transferred into 50 ml conical test tubes. Cells were pelleted bycentrifugation at 2,000×G, for 5 minutes, 21° C. and the cell pellet waswashed with TBS. Then, two volumes of P2 buffer (20 mM HEPES, pH 7.2, 20mM KCl, 1 mM dithiothreitol, 2.5 μg/ml each of leupeptin, pepstatin,aprotinin, 10 μg/ml aPMSF, 0.5 mM EDTA) were added, and the cell pelletwas homogenized on ice with a glass-Teflon Dounce homogenizer. Thehomogenate was sedimented at 8,000×g for 10 minutes at 4° C., and thesupernatant was resedimented at 100,000×g for 60 minutes at 4° C. Theresulting pellet was resuspended to 5 mg/ml in P2 buffer.

rht was used as is, or optionally, was phosphorylated to obtainphospho-rht. Phospho-rht was obtained by combining rht with an MSNextract (prepared as described above) at a concentration of 150 μg/mleach in 20 mM 4-(2-hydroxyethyl)-1-piperazine ethylenesulfonic acid(HEPES), pH 7.2, 4 mM KCl, 5 mM MgCl₂, 1 mM dithiothreitol, 0.5 mMethylenediaminotetra acetic acid (EDTA), 2.5 μg/ml each of aprotinin,leupeptin, and pepstatin, 10 μg/ml 4-amidino-phenyl methylsulfonylfluoride (a PMSF), 1 μM okadaic acid, 2 mM ATP, 1 mM ethylene glycolbis(b-aminoethylether)N,N,N,N′-tetroacetic acid (EGTA), 10 mMphosphocreatine, and 20 μg/ml creatine phosphokinase.CyclicAMP-dependent kinase catalytic subunit (Pierce) may be included at1.67 units per 10 μl. The reaction is initiated by the addition of theATP, and proceeds at 30° C. for 16 hr with agitation. The reaction isstopped by addition of 2 volumes 5 mM EDTA, 20 mM 2-glycerol phosphate,20% glycerol in BBS, pH 8.3. The phosphorylated rht may be used as is,or may be re-purified by boiling for 10 min, centrifugation at 15,000×gfor 10 min at 4° C., and chromatography on Ni-nitriloacetic acid agarosecolumn as described above.

Hypericin stock was prepared at 20 mM in DMSO, and stored at −20° C. Therht or phospho-rht preparation to be treated was diluted in 20 mMNa₂B₄O₇, 100 mM H₃BO₃, 75 mM NaCl, pH 8.3 (BBS) to twice the desiredassay concentration, and mixed 1:1 with 6 μM hypericin in BBS forgreater than 1 hour, at 21° C. in the light (hypericin islight-sensitive). Substituting 2 to 5 μM calphostin C has a similareffect on rht and phospho-rht immunoreactivity.

Both rht and phospho-rht were treated with hypericin and analyzed forreactivity with human autoantibodies in the sandwich assay describedpreviously. Both rht and phospho-rht were reactive with both Alzheimer'sdisease and control sera even without hypericin treatment, but hypericinincreased the signal intensity 2- to 4-fold for rht, and about 100-foldfor phospho-rht (see Table 3). This amplification of signal makespossible the use of these purified proteins as well-defined antigens forcharacterization and quantitation of human autoantibodies. A68 alsoshowed reactivity with these same sera.

TABLE 3 Hypericin Effects on human serum autoantibody reaction with rhtand phospho-rht AD serum Control serum Antigen (Relative Light Units)(Relative Light Units) rht 70.8 72.4 rht + hypericin 317.6 170.6 A6858.9 4.6 no antigen 1.9 1.7 phospho-rht 4.3 2.4 phospho-rht + hypericin366.1 227.0The results depicted in Table 3 confirm the discovery of agents whichcause rht or phosphorylated rht to increase their respective reactivitywith A68 autoantibodies. These are useful antigens for enhanceddetection of human autoantibodies in AD.

Example 8 Increasing the Reactivity of Components of Alzheimer's diseaseAntigen

This example describes methods of treating agents (i.e., whichconstitute components of the Alzheimer's disease antigen, such as tau,phosphorylated tau, and the like) such that their condition followingsuch treatment optimally mirrors the state in which they are present inan A68 antigen preparation according to the invention which issubstantially free of immunoglobulin G. Such treatment thus provides fora method of increasing the ability of these agents to detectautoantibodies that are present in Alzheimer's disease.

For instance, preferably according to the invention, it is possible touse for the detection of Alzheimer's disease autoantibodies recombinanthuman tau (rht) that has been treated so that it will function in asimilar fashion as an Alzheimer's disease antigen. One such methodcomprises phosphorylation of rht, desirably by using a cell extractprepared from neuroblastoma cells (e.g., MSN neuroblastoma cells) thathave been optionally treated with okadaic acid (OKA) which increases thecell extract's ability to hyperphosphorylate rht. Hyperphosphorylatedtau is a major component of Protein A/G treated A68. Thehyperphosphorylated rht produced by such treatment desirably can also beused as an Alzheimer's disease antigen to detect human autoantibodies inserum.

Similarly, rht and/or phosphorylated rht (as detailed above) optimallycan be treated with hypericin (or calphostin C) to produce anAlzheimer's disease antigen suitable to detect autoantibodies diagnosticfor Alzheimer's disease. Thus, hypericin (or calphostin C) treated rhtdesirably can be employed according to the invention as a suitableAlzheimer's disease antigen that can be used to detect autoantibodiesdiagnostic for Alzheimer's disease.

Also, rht optionally can be treated with free fatty acids (FFA),according to Wilson and Binder (Am. J. Path, 150, (6), pp. 2181-95(1997); J. Biol. Chem., 270, (41), pp. 24306-14, (1995)). Suchtreatment, preferably using unsaturated fatty acids (e.g., including butnot limited to oleic or linoleic acids, most preferably arachidonicacid) results in the polymerization of the rht, which polymerizationalso is a characteristic of Protein A/G treated A68. Thus, FFA treatedrht is a suitable Alzheimer's disease antigen that can be used to detectautoantibodies diagnostic for Alzheimer's disease.

Moreover, it will be clear to one skilled in the art that use ofpurified or partially purified kinases which have been associated in theliterature with tau phosphorylation either singly or in combination canproduce hyperphosphorylated rht or hyperphosphorylated isolated normaltau suitable as an Alzheimer's disease antigen to detect autoantibodiesdiagnostic for Alzheimer's disease. Such kinases include, but are notlimited to, PKA, GSK, cdc2, cdc25, casein kinase I and II, MAP kinase,and PHF kinase.

In still another manifestation of the invention, it has been describedby Smith, Sayer, Monnier, Perry, et al. (Trends in Neuroscience, 18,(4), pp. 172-6, (1995); Proc. Natl. Acad. Sci. USA, 91, pp. 5710-14,(1994); Ann. NY Acad. Sci., 738, pp. 447-54, (1994)), that advancedglycation endproducts (AGEs) result in the crosslinking of tau via theMaillard reaction and Amadori rearrangement. There is evidence of thiscrosslinked tau in neurofibrillary tangles which, as previouslyindicated, are comprised of A68/hyperphosphorylated tau. Thus,preferably treatment of rht using the lipid peroxidation product4-hydroxy-2-nonenal (HNE) or other AGEs either alone, or in combination,produces an Alzheimer's disease antigen suitable for the detection ofautoantibodies diagnostic for Alzheimer's disease.

These methods thus can be employed to increase the ability of anAlzheimer's disease antigen (i.e., isolated components of the antigen)to detect autoantibodies that are present in Alzheimer's disease.

Example 9 Purification of Bovine Tau

This Example describes the purification of bovine tau.

Bovine brains are obtained as soon after slaughter as possible andplaced in ice water. All subsequent steps are at 4° C. unless otherwiseindicated. Large blood clots are removed from 600 gms cerebral cortex.The overlying meninges may be removed as well. 2-mercaptoethanol andPMSF is added to 900 ml 0.1 M 1,4-piperazinediethanesulfonic acid(PIPES)-NaOH, pH 6.6, 1 mM EGTA, 1 mM MgSO₄ (PEM) to a finalconcentration of 1 mM each, and the brains are placed therein. Thebrains are homogenized using a Waring blender for 4 seconds at lowspeed, then 4 seconds at medium speed. The homogenate is subjected tocentrifugation at 23,000×g for 90 minutes at 2° C., and the supernatantis carefully collected. GTP is added to the supernatant to a finalconcentration of 1.0 mM. Alternatively, GTP can be added to 0.1 mM, andATP can be added to 2.5 mM. Incubation at 37° C. with gentle swirling isperformed for 30 minutes in a large flask to assemble microtubules. Thesupernatant is then transferred to centrifuge bottles underlayed with 20ml 10% sucrose in PEM containing 1 mM GTP, and subjected tocentrifugation at 37° C. for 45 minutes at 23,000×g. The supernatant isdiscarded, the pellet is resuspended in 75 ml PEM containing 1 mM GTP at0° C., and homogenized with a teflon/glass homogenizer (2 passes at 2000rpm), and incubated on ice for 30 minutes to disassemble themicrotubules. The mixture is then subjected to centrifugation at38,000×g for 30 minutes at 2° C. The supernatant is decanted intopreweighed centrifuge tubes and incubated at 37° C. for 15 minutes torepolymerize the microtubules. The tubes are then subjected tocentrifugation at 38,000×g at 37° C. for 30 minutes. This pelletcontains MAPs and tubulin in purified form. To separate MAPs fromtubulin, the pellet is resuspended in ⅓ volume PEM containing 1 mM GTP,1 M NaCl at 0° C. and the liquefied pellet is loaded at 0.5 ml/min, 4°C., onto a DEAE-sephadex (A-50, Pharmacia) column, 1 ml bed volume/mlpellet, which has been equilibrated in PEM containing 1 mM GTP, 0.25 MNaCl. MAPs elute in the unbound fraction, are termed MAPf. Subsequently,bovine tau is purified further from MAPf on phosphocellulose.

An alternative preparation of tau protein utilizes the first supernatantdescribed above, but instead of assembling the microtubules, thesupernatant is heated to 90° C. for 5 min. It is then subjected tocentrifugation at 23,000×g for 90 min at 4° C. The tau protein issubsequently concentrated and partially purified by anion exchangechromatography on DEAE-cellulose (Whatman DE52) equilibrated in PEM. Thetau protein is eluted with a linear 0-1 M NaCl salt gradient, and storedat −80° C.

Example 10 Reverse ELISA Sandwiches for A68 and Bovine Tau

This Example describes reverse sandwiches for A68 and bovine tau.

Autoantibodies reactive with A68 and bovine tau also can be detected ina sandwich wherein the antigen is immobilized. This is accomplished bycoating Dynex Microlite 1 plates with 3 μg/ml goat anti-mouse Ig(Fc) incoat buffer for 3 hours at 24° C., and subsequently blocking with 288 μl5% non-fat dry milk in BBS for 1 hour at 24° C. For A68 capture,monoclonal Ab PHF1 at 1 μg/ml in casein/TBS is added. For bovine taucapture, taul (Roche) at 1 μg/ml in casein/TBS is added. Both areincubated for 2 hours at 24° C. 1 μl A68 (about 30 ng) or 1 μl MAPf(about 100 ng bovine tau) are added in 5% fetal bovine serum (HyClone)in BBS for 20 hours at 4° C. Alternative antigens (e.g. phosphorylatedrht+/−hypericin) may be utilized. Serum to be tested for autoantibodiesis added to wells at 1:100 dilution in 1% non-fat dry milk, 5% normalgoat serum (Sigma), BBS and incubated 16 hours at 4° C. Boundautoantibody is detected with 0.27 μg/ml horseradishperoxidase-conjugated goat anti-human Ig in casein/TBS for 2 hours at24° C. followed by addition of 90 μl LumiGlo (Kirkegaard and Perry)chemiluminescent substrate.

Chemiluminescence is measured as described above. In between each step,wells are washed with 0.1% Tween-20, tbs. All incubations are 100 μlexcept where indicated.

Example 11 Use of Bovine MAPf and Tau in Conjunction With A68 in WesternBlot Analysis

This Example describes the use of Bovine MAPf in conjunction with A68 inWestern blot analysis of sera.

Bovine MAPf contains, among other things, 70% MAPs 1 & 2, 20% otherMAPs, and 10% Tau MAP isoforms. The current invention, as describedhere, makes use of the six Tau MAP isoforms that migrate in the 40-65 kDrange on a 10% SDS polyacrlyamide gel. The methods for electrophoresis,Western transfer, sera incubation, and detection of bound autoantibodyare identical to the methods described previously in Example 3. Theexception in this Example is the use of alternating lanes of A68 andMAPf for electrophoresis. MAPf is used at the concentration of 1.5 ugtotal protein/lane. Patient sera is incubated with the strips ofnitrocellulose containing purified A68 protein preparation and strips ofnitrocellulose containing MAPf, and bound autoantibodies are thenvisualized as previously described. Bound autoantibodies to purified A68protein preparation and the tau isoforms in MAPf are then quantifiedusing a transmittance densitometer (Bio-Rad GS-670) and MolecularAnalyst Image Analysis Software (Bio-Rad Version 1.1.1). After acquiringan image of the Western blot using the densitometer, the softwareprogram is used to create a one-dimensional profile of the band patternsproduced by the sera on purified A68 protein preparation and tauisoforms of MAPf. The profiles are then background subtracted. Theresulting profiles contain peaks that represent both the width of theband as well as the optical density (OD) of bound antibodies.Integration of the area under the peak provides a numerical measure ofbound antibodies in units of OD×mm.

Two methods of analysis are currently used to assign a diagnosis to eachserum tested. In the first method, the total OD×mm signal from purifiedA68 protein preparation is divided by the total OD×mm signal from thetau isoforms. The sample is assigned the diagnosis of AD, or non-ADbased on this ratio.

The second method takes into account not only optical densitymeasurements, but also the number of MAPf tau isoforms identified by agiven serum. In this method of analysis, if a bovine MAPf tau signal ispresent in conjunction with purified A68 protein preparation, the sampleis assigned a diagnosis of AD if the tau signal contains less than threeisoforms, and a diagnosis of non-AD if the sample identifies 3 or moreisoforms of tau. As well, the sample is classified as non-AD if it lackspurified A68 protein preparation signal, regardless of the number of taubands. Quantification of the MAPf tau signal in this instance takes onthe formula: (sum OD×(n−2)). For this formula, “Sum OD” is the sum ofOD×mm measurements of all tau isoforms and “n” is the total number ofbands present of the six tau MAPf isoforms. Thus, samples that give apurified A68 protein preparation signal and lack a substantial tausignal are termed AD, all other combinations (A68 signal+tau signal, tausignal alone, or absence of both signals) are diagnosed as non-AD.

The use of tau isoforms is not limited here to Bovine tau isoforms foundin MAPf. Other forms of tau protein may be used such as tau purifiedfrom brain and recombinant tau either as a single molecule or as amixture of tau isoforms. Additionally, the invention is not limited toBovine tau. Purified tau or MAP from brains or cultured cells of otherspecies may be used such as human, rodent, or other mammalian sources aswell as preparations from avian and reptiles.

Example 12 Indirect ELISA on Nitrocellulose Plates

This Example describes an indirect ELISA on nitrocellulose plates.

Autoantibodies reactive with purified A68 protein preparation and bovinetau may also be detected in an indirect ELISA assay wherein the antigenis immobilized in a microtiter plate in which the bottom of each well isnitrocellulose. This support allows the antigen to be displayed in amanner which more closely resembles the Western blot than does apolystyrene support. Millipore MHAB plates are pre-wet with BBS for 1minute, then the buffer is drawn through the filter under vacuum.Antigen is applied to the wells in BBS at 0.01 to 10 μl per well (0.3 to300 ng), and allowed to bind for 3 hours at 24° C. In these experiments,the antigen may be purified A68 protein preparation, bovine tau (MAPf),or purified A68 protein preparation analogues such as phosphorylatedrht. The antigen solution is drawn through the filter under vacuum, andthe filters are blocked with 5% non-fat dry milk in BBS for 1.5 hours at24° C. Subsequently, all incubation solutions are removed by a platewasher (Nunc) and washed with 0.1% Tween 20 in TBS rather than bydrawing through the membrane under vacuum. 1% serum is added to wells in100 μl of 1% non-fat dry milk, 5% normal goat serum, BBS, and incubatedfor 16 hours at 4° C. Bound human Ig is detected by addition ofHRP-conjugated goat anti-human Ig in 1% casein/TBS for 2 hours at 24° C.followed by addition of 90 μl LumiGlo (Kirkegaard and Perry)chemiluminescent substrate. Chemiluminescence is measured as describedabove.

All of the references cited herein are hereby incorporated in theirentireties by reference. In particular, the entire text and teachings ofPCT International Application WO 96/20218 is hereby incorporated byreference.

While this invention has been described with an emphasis upon apreferred embodiment, it will be obvious to those of ordinary skill inthe art that variations in the preferred composition and method may beused and that it is intended that the invention may be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications encompassed within the spirit andscope of the invention as defined by the following claims.

1. A protein preparation consisting essentially of an antigen that isimmunologically reactive with a monoclonal antibody produced by thehybridoma cell line identified as ATCC No. HB9205, said preparationbeing substantially free of immunoglobulin G.
 2. The protein preparationof claim 1, wherein said preparation has an amount of immunoglobulin Gthat is equal to or less than about 0.05% of the total protein of saidpreparation.
 3. The protein preparation of claim 1, wherein saidpreparation has an amount of immunoglobulin G that is equal to or lessthan about 0.0015% of the total protein of said preparation.
 4. Theprotein preparation of claim 1, wherein said preparation has less thanabout 500 pg of immunoglobulin G per μg of said antigen.
 5. The proteinpreparation of claim 1, wherein said preparation has less than about 15pg of immunoglobulin G per μg of said antigen.
 6. A protein preparationconsisting essentially of an antigen, which preparation is a diagnosticmarker of Alzheimer's disease, wherein said antigen comprises a majorpolypeptide species that: (a) has an isoelectric point of about 6 inreduced or non-reduced form; (b) binds to an affi-Blue column; (c) is atleast 50% soluble in a solution of 0.01 M sodium phosphate, 0.14 Msodium chloride and 1 mM phenyl methyl sulfonyl fluoride at pH 6.8, andprecipitates in 50% saturated ammonium sulfate at 4° C.; (d) isimmunologically reactive with a monoclonal antibody produced by thehybridoma cell line identified as ATCC No. HB9205; and (e) issubstantially free of immunoglobulin G.
 7. The protein preparation ofclaim 6, wherein said preparation has an amount of immunoglobulin G thatis equal to or less than about 0.05% of the total protein of saidpreparation.
 8. The protein preparation of claim 6, wherein saidpreparation has an amount of immunoglobulin G that is equal to or lessthan about 0.0015% of the total protein of said preparation.
 9. Theprotein preparation of claim 6, wherein said preparation has less thanabout 500 pg of immunoglobulin G per μg of said antigen.
 10. The proteinpreparation of claim 6, wherein said preparation has less than about 15pg of immunoglobulin G per μg of said antigen.
 11. A process forobtaining the protein preparation of claim 1, said process comprising:(a) obtaining a sample of cortical brain tissue containing said antigen;(b) homogenizing said sample in buffer to obtain a homogenate; (c)removing particulate matter from said homogenate; (d) removing saidantigen from said homogenate by contacting the homogenate with anantibody under conditions wherein said antigen and said antibody form anantigen-antibody complex; (e) eluting said antigen from saidantigen-antibody complex; and (f) removing immunoglobulin G from theeluent to obtain said protein preparation.
 12. The process of claim 11,wherein said immunoglobulin G is removed by incubation of said proteinpreparation with: (a) Protein A; (b) Protein G; (c) both Protein A andProtein G; or (d) an immunoglobulin G removal method that issubstantially equivalent to (c).
 13. In an improvement of a process forobtaining a preparation consisting essentially of an antigen that isimmunologically reactive with the monoclonal antibody produced by thehybridoma cell line identified as ATCC No. HB9205, said improvementcomprising removing immunoglobulin G from the antigen preparation toobtain a preparation that is substantially free of immunoglobulin G.14-37. (canceled)